Despite media reports he wants to lead the party, there was "not a snowball's chance in Hades" he would stand for MP if Londoners re-elected him, he said.
That means any ambitions to become Tory leader - or PM - would have to wait.
Mr Johnson has been hotly-tipped as a successor to David Cameron for some time but he added: "I don't think I will do another big job in politics after [being mayor]."
He had used his speech at the party conference in Manchester to declare pride in his record as mayor, praise Londoners for their response to the riots and call for spending on London's transport to be used as a "locomotive" to power the wider UK economy.
Later, he spoke more widely about Conservative policy, telling Newsnight that retaining the 50p top rate of income tax for people earning over £150,000 risked making the UK look "less competitive".
'Looser' EU ties In a fringe event for London's LBC radio, Mr Johnson raised the possibility of a referendum on the UK's relationship with the European Union.
"The British people haven't had a say on Europe since 1975. There hasn't been a vote. If a reasonable question could be framed and put to the people of this country... it is not a bad idea," he said.
However, he added that he was not sure what the outcome would be and that Britons may not opt to leave the EU but simply prefer a "looser" relationship.
There had been suggestions that the conference might have featured dissent from backbenchers on the referendum issue, although none have spoken out publicly.
And Mr Johnson's spokesman later said the mayor had only answered a hypothetical question and did not believe a referendum at this point "would serve any useful purpose".
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